Abstract
DOPA 4, 5-dioxygenase (DOD) is the most important enzyme involved in betalain biosynthesis. The cDNA coding DOD was isolated from common portulaca (Portulaca grandiflora) by cDNA subtraction method (Christinet, et al., Plant Physiol 134: 265-274). It was confirmed by complementation experiments using the particle bombardment that the gene was concerned with betalamic acid synthesis in vivo, but there has been no reports to detecte DOD activity in vitro. In this study, we isolated MjDOD cDNA from four o'clock (Mirabilis jalapa) by PCR method using the degenerate primer based on the conserved amino acid sequence among plant DOD genes. A MjDOD cDNA was cloned into yeast expression vector and introduced into Saccharomyces cerevisiae. A crude extract from the recombinant yeast strains was added to a reaction mixture containing L-DOPA. The color of the reaction mixture turned to yellow and a reaction product in the mixture was betalamic acid confirmed by HPLC.
